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Antimicrobial Agents and Chemotherapy, November 2002, p. 3688-3689, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3688-3689.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Phenotype and Molecular Characterization of the First Inhibitor-Resistant TEM-Derived ß-Lactamase Identified in Portugal


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LETTER
 
Since the description of the first TEM-derived ß-lactamases conferring resistance to clavulanate, a number of these enzymes have emerged in various parts of the world, as profiled by Jacoby and Bush (http://lahey.org/studies/webt.htm).

There is little information available on resistance to ß-lactam-ß-lactamase inhibitor combinations in members of the family Enterobacteriaceae in Portugal (2, 4). Here, we report the first phenotypic and molecular characterization of a Portuguese clinical isolate harboring an inhibitor-resistant TEM-derived ß-lactamase (IRT).

Escherichia coli strain INSRA99 was isolated in January 1999 from the urine of a 20-year-old woman hospitalized at SAMS Hospital in Lisbon. In disk diffusion tests at the National Institute of Health in Lisbon, the strain was resistant to amoxicillin and amoxicillin-clavulanate. Its phenotype and genotype were studied to determine if the clavulanate resistance was due to an inhibitor-resistant TEM-derived ß-lactamase (IRT)-, an AmpC-, or OXA-type-producing mechanism or was due to the hyperproduction of TEM ß-lactamases.

Resistance was cotransferred to E. coli strain HB101. MICs of various ß-lactams, alone or in combination with ß-lactamase inhibitors (Table 1), were determined by the agar dilution method, as described in the 2000 report of the Antibiogram Committee of the French Society for Microbiology 2000-2001 (G. Carret, J. D. Cavallo, H. Chardon, C. Chidiac, P. Choutet, P. Courvalin, H. Dabernat, H. Drugeon, L. Dubreuil, F. Goldstein, V. Jarlier, R. Leclercq, M. H. Nicolas-Chanoine, A. Philippon, C. Quentin, B. Rouveix, J. Sirot, and C.-J. Soussy). Strains producing TEM-1, IRT-2, OXA-1, and AmpC enzymes were used as references.


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  		TABLE 1. MICs of ß-lactam antibiotics for E. coli INSRA99, the transconjugant E. coli HB101-RA99, and other E. coli strains

E. coli INSRA99 showed resistance to amoxicillin and ticarcillin, alone and in combination with ß-lactamase inhibitors, and susceptibility to the other ß-lactams (Table 1). The transconjugant E. coli HB101-RA99 showed less resistance than E. coli INSRA99, being susceptible to ticarcillin-clavulanic acid and to the other ß-lactams. Isoelectric focusing showed a single enzyme with a pI of 5.2.

A 1,092-bp blaTEM DNA fragment obtained by PCR and purified with Qiaquick spin columns (Qiagen, Hilden, Germany) was sequenced as previously described (3) with an ABI377 sequencer (Applied Biosystems, Foster City, Calif.). The sequence revealed a blaTEM-1-derived gene with the nucleotide substitution C929->A (Arg244->Ser). This mutation explains why the pI of INSRA99 ß-lactamase is more acidic than that of TEM-1. We conclude that the enzyme is TEM-30 (IRT-2) (1).

The coding regions of blaINSRA99 and blaHB101-RA99 are similar to that of the parental blaTEM-1B, but 1 nucleotide position in the promoter region, G162->T, differs, corresponding to the P4 promoter (5). The presence of T162 in the Pribnow box of blaINSRA99, blaHB101-RA99 in this study, and blaP20 (3) promoters may contribute to overproduction of the inhibitor-resistant enzymes produced by the corresponding E. coli strains. This promoter mutation was first identified in a blaESBL (6) and has been described in blaIRT genes (3): it was mostly associated with a TEM-2-like framework. However, the blaTEM-1B frameworks of the blaINSRA99 gene and of other blaIRT-2 genes (7) associated with the promoter variation enlarge the molecular diversity and indicate convergent evolution of blaTEM and blaIRT genes.

In this study, the susceptibility profile of E. coli, the pI of the ß-lactamase produced, and the molecular analysis of the gene coding for that enzyme are in agreement and suggest that there is a selective pressure from ß-lactamase inhibitors in Portugal. The worldwide spread of resistance to ß-lactamase inhibitors may impede the use of ß-lactams for effective treatment of bacterial infections.


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Nucleotide sequence accession number.
 
The nucleotide sequence of blaINSRA99 has been deposited in the EMBL Nucleotide Database under accession no. AJ437107.


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ACKNOWLEDGMENTS
 
We thank G. Paul for kindly providing E. coli Guer and P20 and C. Féria for the gift of E. coli FMV347 and FMVAmpC. We thank Sónia Pedro for running the sequence reactions. We also thank the following pharmaceutical companies represented in Portugal for having supplied antibiotics: GlaxoSmithKline Beecham (amoxicillin, ticarcillin, and clavulanic acid), Wyeth Lederle Portugal (piperacillin and tazobactam), Leo Pharmaceutical Products (mecillinam), Lilly Farma (cephalothin), Farma-APS (cefuroxime), Pfizer (cefoperazone), Bristol-Myers Squibb (cefepime and aztreonam), GlaxoWellcome (ceftazidime), Roche Pharmaceuticals (ceftriaxone), and Aventis Pharma (cefotaxime).

This work was supported by grant CFICS/189/99 from the Ministério da Saúde, Portugal.


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REFERENCES
 
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  1. Belaaouaj, A., C. Lapoumeroulie, M. M. Caniça, G. Vedel, P. Névot, R. Krishnamoorthy, and G. Paul. 1994. Nucleotide sequences of the genes coding for the TEM-like ß-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiol. Lett. 120:75-80.[Medline]
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  2. Caniça, M., M. Ferreira, V. Vaz-Pato, E. Ferreira, and Grupo de Estudo Multicêntrico de VigilÂncia da Susceptibilidade aos Antibióticos. 2000. Mecanismos de resistência aos ß-lactÂmicos em estirpes de Escherichia coli de origem clínica. Arq. Med. 14:71.
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  3. Caniça, M. M., C. Y. Lu, R. Krishnamoorthy, and G. C. Paul. 1997. Molecular diversity and evolution of blaTEM genes encoding ß-lactamases resistant to clavulanic acid in clinical E. coli. J. Mol. Evol. 44:57-65.[CrossRef][Medline]
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  4. Féria, C., E. Ferreira, J. D. Correia, J. Gonçalves, and M. Caniça. 2002. Patterns and mechanisms of resistance to ß-lactams and ß-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal. J. Antimicrob. Chemother. 49:77-85.[Abstract/Free Full Text]
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  5. Goussard, S., and P. Courvalin. 1999. Update sequence information for TEM ß-lactamase genes. Antimicrob. Agents Chemother. 43:367-370.[Abstract/Free Full Text]
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Manuela Caniça*
Mónica Ferreira
Eugénia Ferreira
Antibiotic Resistance Unit
Centre of Bacteriology
National Institute of Health Dr. Ricardo Jorge
Av. Padre Cruz
1649-016 Lisboa, Portugal

Luísa Cabral
Laboratory of Microbiology
Hospital SAMS
1800 Lisboa, Portugal

* Phone and fax: 351.217519246
E-mail: manuela.canica{at}insa.min-saude.pt

Antimicrobial Agents and Chemotherapy, November 2002, p. 3688-3689, Vol. 46, No. 11
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.11.3688-3689.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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